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Endocrinology Vol. 140, No. 10 4821-4830
Copyright © 1999 by The Endocrine Society

ARTICLES

Expression of Insulin-Like Growth Factor I Messenger Ribonucleic Acid in Developing Osteophytes in Murine Experimental Osteoarthritis and in Rats Inoculated with Growth Hormone-Secreting Tumor

Ken Okazaki, Seiya Jingushi, Takashi Ikenoue, Ken Urabe, Hiroaki Sakai, Akira Ohtsuru, Kozo Akino, Shunichi Yamashita, Shintaro Nomura and Yukihide Iwamoto

Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3–1-1 Maidashi, Higashi-ku, Fukuoka; the Department of Nature Medicine, Atomic Bomb Disease Institute (A.O., S.Y.), and the Department of Anatomy I, Nagasaki University School of Medicine (K.A.), 1-7-1, Sakamoto, Nagasaki, Japan 852-8501; and the Department of Pathology, Osaka University Medical School (S.N.), 2-2, Yamadaoka Suita, Osaka, Japan 565-0871

Address all correspondence and requests for reprints to: Seiya Jingushi M.D., Ph.D., Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3–1-1 Maidashi, Higashi-ku, Fukuoka, Japan. E-mail: jingushi{at}ortho.med.kyushu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteophytes are one of the characteristic features of osteoarthritis and are often found in acromegalic arthropathy. The aim of this study was to investigate insulin-like growth factor I (IGF-I) involvement in osteophyte formation. One percent collagenase solution was injected into murine knee joints as an osteoarthritis model. In a different animal group, GH-secreting tumor cells were inoculated sc to the rat thigh as an acromegaly model. A series of osteophyte formation was examined histologically. IGF-I messenger RNA was detected using the in situ hybridization method. Type I IGF receptors were detected immunohistochemically. In the osteoarthritis model, osteophyte formation appeared as synovial or perichondral cell proliferation adjacent to the articular cartilage on day 5, followed by cartilage formation on day 7 and endochondral ossification on day 14. In the acromegaly model, synovial or perichondral cell proliferation was observed 4 weeks after inoculation, followed by osteophyte formation at 8 weeks. In both models, IGF-I messenger RNA and type I IGF receptor were coexpressed by proliferating synovial or perichondral cells, proliferating chondrocytes, and osteoblasts within the developing osteophytes. These results suggest that IGF-I regulated the initiation and development of osteophyte formation in both models in an autocrine and/or paracrine fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOARTHRITIS is characterized by a disruption of the equilibrium between synthesis and degradation of matrix molecules in articular cartilage. Osteophyte formation, one of the characteristic features of osteoarthritis joints, includes new cartilage formation and subsequent endochondral ossification at the joint periphery (1). It is recognized as an attempt at repair, with a view to broadening the joint surface and stabilizing the degenerating joints.

In acromegaly, in which circulating GH is increased due to the presence of a GH-secreting pituitary tumor, there is usually significant osteophyte formation similar to that seen in osteoarthritis joints, in addition to articular cartilage thickness within the joints (2, 3, 4). Insulin-like growth factor I (IGF-I) is known to be the main mediator of GH and is synthesized in the liver and epiphyseal cartilage (5, 6, 7, 8, 9, 10, 11, 12). IGF-I is also known to be an important anabolic factor for bone and cartilage metabolisms. IGF-I can stimulate cell proliferation (13, 14) and the synthesis of both proteoglycan (13, 14, 15, 16) and type II collagen (16, 17) in chondrocytes from the articular cartilage or the epiphyseal cartilage. Such findings suggest that IGF-I may play some role in regulating osteophyte formation, in which cartilage formation and endochondral ossification are observed.

It has been reported that a single intraarticular injection of collagenase into a murine knee joint causes osteoarthritic changes (18, 19, 20). Subcutaneous inoculation with GH-secreting pituitary tumor cells in adult rats produced a remarkable elevation of serum GH concentration, bringing about an acromegalic phenotype (21, 22, 23). These findings led us to use these two animal models to investigate IGF-I involvement in the local regulatory mechanism in osteophyte formation. We demonstrated the expression of IGF-I messenger RNA (mRNA) and type I IGF receptors in developing osteophytes in the osteoarthritis model as well as in rats inoculated with GH-secreting tumor cells, and we herein discuss the role of IGF-I in osteophyte formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
An experimental osteoarthritis model
Sodium pentobarbital (0.075 mg/g BW) was injected ip into 10-week-old male C57BL10 mice, and 10 µl 1.0% (wt/vol) bacterial collagenase solution (248 U/mg; Worthington Biochemical Corp., Freehold, NJ) were injected once into the left knee joints with a 30-gauge needle. The same volume of PBS was similarly injected into the contralateral knee joints as a control. The mice were permitted to bear their full weight and were allowed unrestricted activity after waking from the anesthesia. Thereafter, they were killed 5, 7, or 14 days after the injection, and their whole knee joints were harvested for further analysis. This experiment was reviewed by the committee of the ethics on animal experiments at the Faculty of Medicine, Kyushu University, and was carried out under the control of the Guidelines for Animal Experiments at the Faculty of Medicine, Kyushu University, and The Law (No. 105) and Notification (No. 6) of the Government.

An experimental acromegalic arthropathy model
The rat malignant pituitary somatotroph cell line mGH3 cells was used (24). Ten 6-week-old female Wister-Furth rats were injected sc in their left thigh with 1.0 x 106 mGH3 cells suspended in 0.5 ml Ham’s F-10 medium containing penicillin (50 U/ml) and streptomycin (50 µg/ml). The mGH3-bearing rats were killed 4 or 8 weeks after inoculation. Age-matched untreated rats were used as controls. At the time of death, their body weights were measured. At the same time, serum GH concentrations were analyzed by the RIA method using the rat GH 125I assay system (Amersham International, Aylesbury, UK). Their left or right knee joints were harvested for histological examination, immunohistochemistry, and in situ hybridization.

Histological examination
The specimens were washed with phosphate buffer containing 0.1% diethylpyrocarbonate before being fixed in 4% paraformaldehyde overnight at 4 C. After being decalcified in 20% EDTA, they were embedded in paraffin. Serial frontal sections with a thickness of 5 µm were prepared for histological examination, in situ hybridization, and immunohistochemistry. The sections were stained with safranin-O for histological examinations. Two orthopedic doctors who knew nothing about the protocol of the experiment evaluated the specimens histologically. With regard to the features of osteophyte formation, we evaluated multilayers of synovial cells or perichondral cells (five or more layers of these cells adjacent to the articular cartilage), cartilage tissue formation (cartilaginous nodules that consisted of matrix and round-shape chondrocytes appeared adjacent to the articular cartilage), and endochondral ossification (primary spongiosa formation associated with vascular invasion into the cartilaginous nodules).

In situ hybridization
Digoxigenin-11-UTP-labeled single strand RNA probes were prepared using the digoxigenin RNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. A full-length (707-bp) fragment of mouse prepro-IGF-IA complementary DNA (25) and a 600-bp fragment of rat type II procollagen complementary DNA (26) were used to generate antisense and sense probes. The in situ hybridization technique was carried out as described by Nomura (27, 28). Deparaffinized sections were digested with 2 µg/ml proteinase K (Sigma Chemical Co., St. Louis, MO) for 15 min at 37 C, followed by 0.2 N HCl for 10 min to inactivate internal alkaline phosphatase. Hybridization was performed at 50 C overnight in a solution containing 50% formamide, 10 mM Tris-HCl (pH 7.6), 200 µg/ml yeast transfer RNA, 1 x Denhart’s solution, 600 mM NaCl, 10% dextran sulfate, 0.25% SDS, and 1 mM EDTA. After hybridization, the sections were washed with 50% formamide and 2 x saline-sodium citrate for 30 min at 50 C and digested with 10 µg/ml ribonuclease A (Roche Molecular Biochemicals) for 30 min at 37 C. For detection of the hybridized digoxigenin-labeled RNA probe, a digoxigenin nucleic acid detection kit (Roche Molecular Biochemicals) was used.

Immunohistochemistry for proliferating cell nuclear antigen (PCNA) and type I IGF receptor
To detect proliferating cells and type I IGF receptors, we performed immunohistochemistry as described previously (29, 30). Deparaffinized sections were incubated in 0.5% hydrogen peroxide in methanol for 60 min to block endogenous peroxidase activity, followed by 1 mg/ml hyaluronidase (Sigma Chemical Co.) in 0.2 M sodium acetate buffer containing 0.5 M NaCl (pH 5.5) for 30 min at 37 C. After a blocking solution (130 µl of normal serum in Tris-buffered saline containing 0.5% BSA), the sections were incubated with anti-PCNA monoclonal antibodies, PC10 (Dakopatts, Copenhagen, Denmark), or anti-type I IGF receptor polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted to 1:200 and 1:100, respectively. Purified mouse and rabbit IgG (Santa Cruz Biotechnology, Inc.) were used as control primary antibodies. A subsequent reaction was performed using a Vectastain avidin-biotin-peroxidase complex kit (Vector Laboratories, Inc., Burlingame, CA). The sections were reacted with diaminobenzidine solution and then counterstained with hematoxylin or methyl green.

Statistics
Data for body weight and serum GH concentrations in mGH3-bearing rats are presented as the mean ± SD, and differences were determined using a nonparametric test (Mann-Whitney’s U test).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Osteophyte formation in the murine collagenase-injected joints (Fig. 1Go)
In the control joints, there were no histological changes on day 5, 7, or 14. In the collagenase-injected joints, multilayered synovial cells or perichondral cells were observed adjacent to the articular cartilage or at the insertion site of intraarticular ligaments on day 5. On day 7, cartilage tissue appeared. Then, endochondral ossification occurred, and subsequently, composite tissue comprising cartilage and bone, so-called osteophytes, was observed on day 14. These histological changes were reproducible in the osteoarthritis model joints (Table 1Go). Additionally, synovial hyperplasia was observed, especially on days 7 and 14. Loss of safranin-O staining and destruction of articular cartilage were not observed on day 7, but were observed in all joints on day 14 in the collagenase-injected joints.



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  		Figure 1. Osteophyte formation in murine osteoarthritis model. A–C, Control joints 7 days after an injection with PBS only. D–L, Collagenase-injected joints: D–F, 5 days; G–I, 7 days; J–L, 14 days after collagenase injection. A, D, G, and J, Low magnified views of the knee joints; safranin-O staining. B, E, H, and K, High magnified views; safranin-O staining. C, F, I, and L, Immunohistochemistry for PCNA. PCNA-positive cells are stained brown (arrowheads). Bars, 100 µm. EC, Epiphyseal cartilage; AC, articular cartilage. The control joints showed no histological changes and very little immunoreactivity for PCNA. In the collagenase-injected joints, multilayered synovial cells or perichondral cells were observed adjacent to the articular cartilage on day 5, and they were PCNA positive. Cartilage tissue was formed on day 7, and endochondral ossification occurred on day 14. The loss of safranin-O staining in articular cartilage was not observed on day 7; however, the destruction of articular cartilage was observed on day 14.

 

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  		Table 1. Histological appearance during osteophyte formation in murine experimental osteoarthritis

 
Immunohistochemistry for PCNA showed that there were many positive cells in the multilayered synovial cells and perichondral cells. Proliferating chondrocytes within the osteophytes were PCNA positive on days 7 and 14, whereas the hypertrophic chondrocytes were not. There were very few PCNA-positive cells in the control joints.

In situ hybridization for type II procollagen mRNA showed that the signals were detected in the proliferating chondrocytes in the developing osteophytes on days 7 and 14, but not in the multilayered synovial cells or perichondral cells. There were very few positive cells in the hypertrophic chondrocytes (Fig. 2Go).



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  		Figure 2. In situ hybridization for IGF-I and type II procollagen in developing osteophytes in murine osteoarthritis model. A–C, Control joints 7 days after a PBS injection. D–L, Collagenase-injected joints: D–F, 5 days; G–I, 7 days; J–L, 14 days after a collagenase injection. A, D, G, and J, Using IGF-I antisense probe. B, E, H, and K, Using type II procollagen antisense probe. C, F, I, and L, Using IGF-I sense probe as each negative control. Bar in J, 100 µm. In the control joints, no IGF-I was detected, and type II procollagen was detected just inside the epiphyseal chondrocytes (B, arrow). Type II procollagen was detected in proliferating chondrocytes in developing osteophytes on days 7 and 14 (H and K, arrows). IGF-I was detected in the multilayered perichondral cells and synovial cells on days 5 (D, arrow) and 7 and in the proliferating chondrocytes on days 7 (G, arrow) and 14 (J, arrow). Additionally, osteoblasts in the endochondral ossification front expressed IGF-I on day 14 (J, arrow).

 
Expression of IGF-I mRNA and type I IGF receptor during osteophyte formation in the murine collagenase-injected joints (Figs. 2Go and 3Go)
In the control joints, there were no signals for IGF-I mRNA in the synovial cells or perichondral cells adjacent to the articular cartilage or in the chondrocytes within the articular cartilage or epiphyseal cartilage. In the collagenase-injected joints, there were many signals for IGF-I mRNA in the multilayered synovial cells and perichondral cells adjacent to the articular cartilage on day 5. IGF-I mRNA was also expressed by proliferating chondrocytes in the osteophytes on days 7 and 14 and by the osteoblasts at the endochondral ossification front in the osteophytes on day 14. There were very few positive cells in the hypertrophic chondrocytes on day 14.



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  		Figure 3. Immunohistochemistry for type I IGF receptor in a murine experimental osteoarthritis model. A, A control joint 7 days after PBS-injection. B–E, Collagenase-injected joints: B, 5 days; C, 7 days; D, 14 days after collagenase injection. E, Negative control using normal rabbit IgG 14 days after collagenase injection. Bar, 100 µm. Immunoreactivities for type I IGF receptor were observed in articular chondrocytes and epiphyseal chondrocytes in the control joints (A, arrowheads). In developing osteophytes, IGF receptors were detected in multilayered synovial cells or perichondral cells on day 5 and in proliferating chondrocytes on days 7 and 14 (arrowheads). At the endochondral ossification front on day 14, osteoblasts were also IGF receptor-positive (arrowhead).

 
Type I IGF receptors were detected in articular chondrocytes and epiphyseal chondrocytes, but were scarcely detected in synovial cells or perichondral cells in the control joints. In the collagenase-injected joints, IGF receptors were detected in the proliferating synovial cells or perichondral cells on day 5 and in the proliferating chondrocytes on days 7 and 14. Additionally, osteoblasts at the endochondral ossification front were also positive for IGF receptors on day 14. As normal rabbit IgG was used as a negative control, no immunostaining was obtained (Fig. 3Go).

Osteophyte formation in the mGH3-bearing rat (Fig. 4Go)
Four weeks after inoculation, there was a tendency for body weight and GH concentration in the mGH3-bearing rats to be greater than those in the control rats, although these differences were not statistically significant. Eight weeks after inoculation, body weight as well as serum GH concentration in the mGH3-bearing rats had increased significantly compared with those in the control rats (Table 2Go).



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  		Figure 4. Osteophyte formation in the knee joints in the rat acromegaly model. Four weeks after inoculation in the mGH3-bearing rats (A and E) and the control rats (B and F). Eight weeks after inoculation in the mGH3-bearing rats (C and G) and the control rats (D and H). A–D, Safranin-O staining. E–H, Immunohistochemistry for PCNA. Bars, 100 µm. EC, Epiphyseal cartilage; AC, articular cartilage. Four weeks after inoculation, many PCNA-positive cells were observed in the multilayered synovial cells and perichondral cells (A, arrow; E, arrowheads). Eight weeks after inoculation, a large osteophyte had formed adjacent to the articular cartilage (C, arrows). Many PCNA-positive cells were observed in the superficial layer of the osteophyte (G, arrowheads). Very few PCNA-positive cells were detected in the control rats (F and H, arrowheads). The articular cartilage demonstrated no degenerative changes in the mGH3-bearing rats or the control rats.

 

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  		Table 2. Body weight and serum GH concentration of the mGH3-bearing rats and the control rats

 
Four weeks after inoculation, multilayered synovial cells and perichondral cells were observed adjacent to the articular cartilage in most of the mGH3-bearing rats (Table 3Go and Fig. 4Go). Immunohistochemistry for PCNA shows that positive cells were detected in the multilayered synovial cells and perichondral cells in the joints of these mGH3-bearing rats, but very few positive cells were detected in the control rats. Eight weeks after inoculation, bone and cartilage tissue appeared adjacent to the articular cartilage in all mGH3-bearing rats. PCNA-positive cells were detected in the proliferating chondrocytes and osteoblasts in the superficial layer of the osteophytes. However, no significant changes were observed within the articular cartilage itself upon histological examination or with immunohistochemistry for PCNA compared with the control rats. These findings were similarly observed in the right knees, contralateral to the limbs inoculated with mGH3 cells (data not shown).


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  		Table 3. Histological appearance during osteophyte formation in the mGH3-bearing rats

 
Expression of IGF-I mRNA and type I IGF receptor during osteophyte formation in mGH3-bearing rats (Figs. 5Go and 6Go)
In situ hybridization for IGF-I mRNA showed that IGF-I mRNA was expressed by the multilayered synovial cells and perichondral cells adjacent to the articular cartilage in most of the knee joints of the mGH3-bearing rats 4 weeks after inoculation (Fig. 5Go). Eight weeks after inoculation, IGF-I mRNA was expressed by the proliferating chondrocytes and by the osteoblasts at the endochondral ossification front of the osteophytes in all the mGH3-bearing rats. The signals for IGF-I mRNA were also detected in epiphyseal chondrocytes in the mGH3-bearing rats, but not in the articular chondrocytes. In the control rats, IGF-I mRNA was not detected at either 4 or 8 weeks. However, type II procollagen mRNA was detected in epiphyseal chondrocytes (data not shown).



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  		Figure 5. IGF-I expression of the knee joint in the mGH3-bearing rats (A and C) and the control rats (B and D). Serial sections in Fig. 4Go are shown. Bar, 100 µm. Signals for IGF-I mRNA were observed in the proliferating perichondral cells and synovial cells as well as in the epiphyseal chondrocytes 4 weeks after inoculation (A, arrows) and in the proliferating chondrocytes and osteoblasts within the osteophytes 8 weeks after inoculation (C, arrows). There were no signals in the control rats.

 


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  		Figure 6. Immunohistochemistry for type I IGF receptor in mGH3-bearing rats (A and C) and in the control rats (B and D). Serial sections in Fig. 4Go are shown. Bar, 100 µm. Immunoreactivities for type I IGF receptor were detected in the articular chondrocytes, epiphyseal chondrocytes, and osteoblasts in the bone marrow in the control rats (B and D, arrowheads) and mGH3-bearing rats. Additionally, there were many IGF receptor-positive cells in the multilayered synovial cells and perichondral cells in mGH3-bearing rats 4 weeks after inoculation (A, arrowheads) and in the proliferating chondrocytes and osteoblasts within the osteophytes 8 weeks after inoculation (C, arrowheads).

 
Type I IGF receptors were detected in the articular chondrocytes in the mGH3-bearing rats and the control rats. Additionally, many IGF receptor-positive cells were detected in proliferating synovial cells or perichondral cells in the mGH3-bearing rats 4 weeks after inoculation. Eight weeks after inoculation, the receptors were detected in the proliferating chondrocytes and the osteoblasts within the osteophytes as well as in the cells around the blood vessels (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used two animal models to investigate IGF-I involvement in osteophyte formation. Van der Kraan et al. reported that a single intraarticular injection with collagenase results in damage to type I collagen-containing joint structures, such as the capsule and ligaments, leading to joint instability, which, in turn, causes osteoarthritic changes (18, 19). On the other hand, sc inoculation with GH-secreting pituitary tumor cells elevates serum GH and IGF-I levels (21, 22, 23). It also has the possibility of stimulating local IGF-I expression at the joint periphery, which leads to osteophyte formation without instability, very similar to acromegalic arthropathy.

A series of osteophyte formation could be observed in the collagenase-injected osteoarthritis model. Proliferation of synovial and perichondral cells was seen adjacent to the articular cartilage on day 5. Cartilage formation was seen at the joint periphery or the insertion site of intraarticular ligaments on day 7. Then, on day 14, endochondral ossification was seen in the cartilage tissue. Although injected collagenase may have influenced type II collagen in the articular cartilage, no changes in the articular cartilage, including the loss of safranin-O staining, were seen histologically on day 7 in this study. Destruction of the articular cartilage was first observed on day 14. This indicates that the destruction of articular cartilage was not due to the collagenase itself, but to joint instability caused by damage to the ligaments or capsule, as described previously (18, 19, 20). In the early phase of osteophyte formation, IGF-I mRNA was expressed by proliferating synovial cells and perichondral cells, which are presumably chondroprogenitor cells. As cartilage tissues were formed, IGF-I was expressed by proliferating chondrocytes that coexpressed type II procollagen mRNA. Then as they differentiated to hypertrophic chondrocytes, the expression of IGF-I mRNA was undetectable. Most IGF-I-expressing cells were PCNA positive in immunohistochemistry, acquiring the type II procollagen message later. Furthermore, type I IGF receptors were detected in these IGF-I-expressing cells. These findings suggest that IGF-I may stimulate both cell proliferation and early differentiation in chondrogenesis during osteophyte formation in an autocrine and/or paracrine fashion. Additionally, IGF-I mRNA was detected in the osteoblasts during endochondral bone formation, and the osteoblasts were positive for PCNA and IGF receptor. IGF-I is known to be one of the anabolic factors for osteoblasts (31), but may also act as a mitogen for them. Recently, it has been reported that a larger amount of IGF-binding proteins (IGFBPs) are synthesized within the osteoarthritis joints. The IGFBPs are considered to be involved in the pathogenesis of osteoarthritis by modulating the IGF-I actions (32, 33). It suggests that IGFBPs may also influence IGF-I actions during osteophyte formation.

The mGH3 pituitary tumor is derived from lymph node metastasis of the rat pituitary somatotroph cell line (GH3) (24). The body weight of the mGH3-bearing rats had increased significantly to approximately 1.5-fold that of the control rats 8 weeks after inoculation in our study. Circulating GH levels had also significantly increased 60- to 600-fold in the mGH3-bearing rats. Yamashita et al. and Imamura et al. previously reported similar results using GH3 cells (21, 22). Four weeks after inoculation in the current study, there were no statistically significant differences in body weight or serum GH concentration between the mGH3-bearing rats and the control rats. There were two rats in which the GH level was similar to the control value. This explains why there were no significant differences regarding the average serum GH level at 4 weeks. The early growth rate of the inoculated tumors varied depending upon the rats and could perhaps have been influenced by the individual conditions of the host rats with regard to tumor cell growth. In the rats that had a relatively high serum GH concentration, histological changes, including perichondral or synovial cell proliferation adjacent to the articular cartilage, and PCNA-positive cells were observed in the joints. On the contrary, in the knee joints of the rats that had a similar GH level as the control rats, such histological changes were not observed. Eight weeks after inoculation, obvious bone and cartilage tissues were seen at the joint periphery in all the mGH3-bearing rats. These osteophytes closely resembled those observed in the osteoarthritis model. The increased body weight may have contributed to induce osteoarthritic changes in the articular cartilage followed by osteophyte formation. However, in this acromegalic arthropathy model, osteophyte formation was observed without the degenerative changes in the articular cartilage that are commonly seen in the osteoarthritis joints. This evidence suggests that other factors seemed to be directly involved in the osteophyte formation rather than simply being a secondary feature following osteoarthritic changes. Both IGF-I mRNA and type I IGF receptors were detected in the proliferating synovial cells and perichondral cells at 4 weeks and in the proliferating chondrocytes and the osteoblasts within the osteophytes at 8 weeks. The cells around the blood vessels were also positive for IGF-I mRNA, IGF receptor and PCNA. These data suggest that the elevation of circulating GH stimulated IGF-I expression in the synovial cells and perichondral cells adjacent to the articular cartilage, which led to the initiation of osteophyte formation. In the mGH3-bearing rats, serum IGF-I probably increased due to the elevation of circulating GH, and this may also have contributed to an increase in IGF-I concentration in the joints, which also functioned to stimulate the development of osteophytes. Such a distinctive stimulation mechanism by IGF-I may be the reason why the histological appearance of osteophytes in the joints of the mGH3-bearing rats was not identical to that of osteophytes in the murine collagenase-injected joints, although both initially demonstrated synovial and perichondral cell proliferation before subsequent cartilage and bone formation. Additionally, the histological changes, including osteophyte formation in the mGH3-bearing rats, were relatively mild compared with those seen in the osteoarthritis model. This may have been due to the relatively short duration of GH excess.

IGF-I is known to be an anabolic factor in bone and cartilage metabolism. It has been reported previously that IGF-I could stimulate cell proliferation and synthesis of proteoglycan or type II collagen in chondrocyte culture or in organ culture of articular cartilage or epiphyseal cartilage (13, 14, 15, 16, 17). IGF-I is expressed by mesenchymal cells or chondrocytes in the epiphyseal cartilage or fracture callus (9, 10, 34, 35). In the epiphyseal cartilage, it is suspected that IGF-I mediates GH action in longitudinal bone growth (5, 6, 7, 8, 9, 10, 11, 12). In our study, IGF-I mRNA was not detected in the epiphyseal cartilage or articular cartilage in the control group of rats and mice. It is suspected that IGF-I expression may have been too weak to be detected in chondrocytes or osteoblasts at the epiphyseal cartilage because of the relatively high age of the animals used in this study (10–14 weeks). Additionally, it was reported recently that IGF-I mRNA expression is not detected in the epiphyseal cartilage of rats or mice at any stage of development from embryo through 5 weeks postnatally (36, 37), contrary to a previous report described by Nilsson et al.(10). In articular chondrocytes, IGF-I mRNA is reported to be barely detectable in normal human (38) and rat joints (39). This evidence may be a further reason why no IGF-I mRNA was detected in the control joints. In the mGH3-bearing rats, the expression of IGF-I mRNA was detected in epiphyseal chondrocytes. This suggests that the IGF-I expression in epiphyseal cartilage was stimulated by excessive circulating GH.

Type I IGF receptors were detected in articular chondrocytes and epiphyseal chondrocytes in the control joints as well as in the osteoarthritis model and the acromegaly model. The receptors seemed to be spontaneously expressed by these cells. Previous studies have reported that mRNA and the protein of this receptor are detected in the normal articular cartilage of humans (33, 38, 40) and mice (41). In the synovial cells or perichondral cells in this study, type I IGF receptors were barely detected in the control joints, but were detected in both the osteoarthritis model and the acromegaly model. This suggests that the expression of this receptor was stimulated in these cells and maintained until they differentiated into proliferating chondrocytes during osteophyte formation. It is reported that IGF-I stimulates the expression of the IGF receptor in cultured rat epiphyseal chondrocytes (42), although there has been less information regarding its expression in synovial cells or perichondral cells. It is speculated that the IGF-I produced may stimulate the expression of the IGF receptor in this osteoarthritis model and acromegalic arthropathy model. Additionally, it is also reported that interleukin-1ß and PGF2{alpha} stimulate the expression of the IGF receptor in cultured rat articular chondrocytes and MC3T3-E1 cells, respectively (39, 43). IGF-I is also reported to be stimulated by such an inflammatory factor, including PGE2 in cultured chondrocytes (44). These inflammatory factors may be involved in the stimulation of the expression of the IGF receptor and IGF-I mRNA, particularly in the osteoarthritis model.

This is the first report regarding IGF-I expression in a series of developing osteophytes, although a study concerning the expression of IGF-I in human osteophytes has been reported previously (45). We detected IGF-I and IGF receptor expression in the osteophytes of joints in an osteoarthritis model as well as in an acromegaly model, suggesting that IGF-I is involved in osteophyte formation in an autocrine and/or paracrine fashion. The distribution and timing of IGF-I and IGF receptor expression were similar in both models, and the regulatory mechanisms of osteophyte formation by IGF-I may be identical. IGF-I is thought to be an important regulatory factor for the initiation and development of osteophyte formation.


   Acknowledgments
 
We thank Dr. T. Shuto for special comments concerning this paper. The English used in this manuscript was revised by Miss K. Miller (Royal English Language Centre, Fukuoka, Japan).

Received February 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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